Nucleic acid amplification techniques are often employed in nucleic acid-based assays used for analyte detection, sensing, forensic and diagnostic applications, genome sequencing, whole-genome amplification, and the like. These applications often require nucleic acid amplification techniques having high specificity, sensitivity, accuracy, and robustness. The amplification of nucleic acids is particularly important when the starting nucleic acid material is present in small amounts.
Various techniques are currently used to amplify nucleic acids, some of which start with only a few molecules of nucleic acid material. These techniques include but are not limited to polymerase chain reaction (PCR), ligase chain reaction (LCR), self-sustained sequence replication (3SR), nucleic acid sequence based amplification (NASBA), strand displacement amplification (SDA), multiple displacement amplification (MDA) and rolling circle amplification (RCA). Several methods are currently available for reverse transcription of RNA to DNA, followed by amplification of the DNA. The current methods of reverse transcription of RNA to DNA and the subsequent amplification of the DNA involves two steps and under different conditions.
In polymerase chain reaction (PCR), a template DNA, a pair of primers, and a DNA polymerase are combined and subjected to repeat temperature cycling that permits melting, annealing, and elongation steps. The melting or denaturation step typically occurs at a high temperature, limiting the choice of polymerases to thermophilic polymerases and the step may further increase the equipment requirements. Alternative amplification methods using strand displacement DNA polymerase, inosine-containing primers, and Endonuclease V under isothermal conditions thereby eliminate the requirement for thermal cycling of the reaction to achieve nucleic acid amplification and the need for polymerase that is thermally stable; able to withstand repeated heating at high temperatures. The Endonuclease V, alternatively referred to as endo V or inosine 3′ endonuclease, is a DNA repair enzyme that recognizes DNA containing nucleotides with deaminated or otherwise modified bases such as inosine. Endonuclease V cleaves the second (or third) phosphodiester bond 3′ to the inosine in the same strand, leaving a nick with a 3′-hydroxyl and 5′-phosphate. In the amplification reaction, DNA polymerases add nucleotides to the 3′ end of a pre-existing DNA strand resulting in 5′→3′ elongation in a template-directed fashion to create a complementary strand. This extension reaction can include displacement of a pre-existing strand, resulting in a net increase in the number of copies of the DNA strand.
Amplification of an RNA template is required in, for example, RNA expression profiling. In this technique, the relative concentration of RNA molecules in a biological sample is determined. Some RNA molecules in a biological sample may be present in relatively low concentrations, such that there is a need to amplify the RNA prior to analysis in order to allow robust detection and analysis. Amplification of mRNA by PCR leads to RNA production of different molecular species at different rates and can provide ambiguous results, making RNA quantification difficult. In addition, the analysis requires at least three steps and different conditions. Currently available RNA amplification techniques suffer from high background noise, which may result from non-specific amplification reactions.
Better methods and compositions for amplification of DNA from an RNA template are desirable. Such methods and compositions should ideally be more robust, highly sensitive and reproducible.